Toxicology 186 (2003) 217
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www.elsevier.com/locate/toxicol

Violacein and its b-cyclodextrin complexes induce apoptosis

and differentiation in HL60 cells
Patrcia S. Melo a, Giselle Z. Justo b, Mariangela B.M. de Azevedo c,
Nelson Duran c,d, Marcela Haun a,*
a

Departamento de Bioqumica, Instituto de Biologia, Universidade Estadual de Campinas, C.P. 6109, CEP 13083-970 Campinas,
Sao Paulo, Brazil
b
Departamento de Farmacologia, Faculdade de Ciencias Medicas, Universidade Estadual de Campinas, C.P. 6111, CEP 13083-970
Campinas, Sao Paulo, Brazil
c
Biological Chemistry Laboratory, Instituto de Qumica, Universidade Estadual de Campinas, C.P. 6184, CEP 13083-970 Campinas,
Sao Paulo, Brazil
d
Nucleo de Ciencias Ambientais, Universidade Mogi das Cruzes, CEP 08790-911 Mogi das Cruzes, Sao Paulo, Brazil
Received 28 October 2002; received in revised form 9 December 2002; accepted 15 December 2002

Abstract
Violacein, a pigment isolated from Chromobacterium violaceum , has been reported to have multiple biological
activities including in vitro antitumor effects. Certain anticancer agents are known to induce apoptosis in human tumor
cell lines. In this work, our aim was to investigate the effectiveness of violacein/b-cyclodextrin (b-CD)-containing
systems to produce lethal effects in the human promyelocytic leukemia cell line HL60. Using the MTT tetrazolium
reduction test, IC50 for the inclusion complexes (1:1 and 1:2 violacein:b-CD molar ratios) and violacein alone were less
than 1 mM. Violacein and violacein/b-CD complexes were able to induce NBT reduction. Moreover, by using the
Feulgen reaction, all the compounds were found to trigger apoptosis in HL60 cells, inducing around 35% of DNA
fragmentation, as analyzed through the diphenylamine assay. In addition, caspases seem to play an important role in
the activation of the executioner phase of apoptosis induced by violacein and its derivatives.
# 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Violacein; HL60 cells; b-Cyclodextrin; Inclusion complex; Differentiation; Apoptosis

1. Introduction
Cancer cells exhibit a defect in their capacity to
mature to non-replicating adult cells, thereby

* Corresponding author. Tel.: /55-19-3788-6150; fax: /5519-3788-6129.

E-mail address: marcelah@unicamp.br (M. Haun).

remaining in a highly proliferative state and outgrowing their normal cellular counterparts. This
permits an alternative approach to the cytodestruction of cancer cells by conventional antineoplastic agents to be envisioned, which involves the
induction of mortality by terminal differentiation
and triggering of the apoptosis process (Uzunoglu
et al., 1999). In this way, therapeutics that

0300-483X/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0300-483X(02)00751-5

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modulate apoptosis provide a new opportunity for

the treatment of numerous diseases including
cancer (Israels and Israels, 1999). Morphologically, in cells undergoing apoptosis there is ruffling, blebbing, and condensation of the plasma
and nuclear membranes, and subsequently aggregation of nuclear chromatin. Mitochondria and
ribosomes retain their gross structure and at least
partial function (Pal et al., 2001). The apoptotic
mode involves an active participation of the
affected cells in their self-destruction cascade that
culminates in DNA degradation via endonuclease
activation, nuclear disintegration, and formation
of apoptotic bodies that are rapidly ingested by
adjacent macrophages or other neighboring phagocytic cells (Rich et al., 2000).
Violacein, a pigment isolated from Chromobacterium violaceum in the Amazon river, Brazil, has
been reported to have multiple biological activities
such as a potential cytotoxic effect and induction
of apoptosis in normal cell cultures (Melo et al.,
2000). We also found that violacein is effective
against a panel of neoplastic cell lines, including
leukemic lineages, suggesting a promising clinical
application in cancer disease (Duran and Menck,
2001). Poor solubility of violacein in water, however, may cause low biological activity in vivo. We
have recently shown that modification in the
physicochemical properties of violacein is achieved
by the preparation of inclusion complexes with bcyclodextrin (b-CD) (de Azevedo et al., 2000a,b).
Natural cyclodextrins (CDs) constitute a family of
cyclic oligosaccharides comprising repetitive 6, 7,
or 8 glucose units (a-, b-, and g-CD, respectively).
The inside of the molecule forms an hydrophobic
cavity, while the outer surface is hydrophilic,
enabling it to act as a host for a wide variety of
lipophilic drugs and components (Zhao et al.,
1996). By complexation, b-CDs can increase the
solubility, stability, bioavailability, and the cell
absorption of the guest molecule (Nicolazzi et al.,
2001).
This study was designed to evaluate the effectiveness of b-CD-containing systems to produce
lethal effects in the human promyelocytic leukemia
cell line HL60. We also investigated the role of
violacein and its b-CD complexes on HL60 cell
differentiation and apoptosis induction.

2. Material and methods

2.1. Violacein
Violacein (3-(1,2-dihydro-5-(5-hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ilydene)-1,3-dihydro2H-indol-2-one) (Fig. 1) was isolated from C.
violaceum and purified as previously described
(Rettori and Duran, 1998).
Violacein/b-CD inclusion complexes were prepared according to de Azevedo et al. (2000a,b).
Briefly, equimolar amounts of violacein and b-CD
(1:1 and 1:2 molar ratios) were dissolved in
acetone and water, respectively, and mixed together. The solvents were evaporated in a vacuum
oven at 50 8C, until complete drying was obtained
and their physicochemical properties were analyzed as previously described (de Azevedo et al.,
2000a,b).
2.2. Cell culture
Human leukemia cells (HL60) were cultured in
suspension in RPMI 1640 medium (Sigma Chemical Co., MO) supplemented with 10% fetal bovine
serum, 100 U/ml of penicillin, and 100 mg/ml of
streptomycin in a humidified atmosphere at 37 8C
in 5% CO2. Cells were seeded (3 /105 cells/ml) in
96-wells and 35-mm plates, and incubated with
different concentrations of b-CD, violacein, violacein/b-CD (1:1) or violacein/b-CD (1:2) for 72 h.
The compounds were firstly dissolved in dimethyl
sulfoxide (DMSO) and then in supplemented
medium. The final concentration of DMSO in
the test medium and controls was 0.1%. Cell
viability was determined by the MTT reduction
test as described elsewhere (Melo et al., 2001).
Each concentration was tested in three different

Fig. 1. Violacein structure.

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experiments run in four replicates. b-CD alone did

not exert any effect on cellular viability in our
experimental conditions, and thus appropriate
controls with free b-CD solutions, corresponding
to equal concentrations of those present in the
respective violacein/b-CD samples, were carried
out and used to study cellular differentiation and
apoptosis induction by violacein/b-CD inclusion
complexes.
2.3. Assay of differentiation
Superoxide production was assayed colorimetrically by the nitro blue tetrazolium (NBT)reducing activity as reported by Kohroki et al.
(1998). After 72 h of treatment with the compounds, the cells were counted and the viability
was assessed by the trypan blue exclusion test.
Viable cells (2 /106 cells/ml) were mixed with an
equal volume of freshly prepared solution containing 200 ng/ml phorbol-12-myristate-13-acetate
(Sigma) and 2 mg/ml NBT (Sigma), and incubated
for 30 min at 37 8C. The reaction was stopped by
adding 5 M HCl (1 M final concentration). The
suspension was centrifuged and the medium
discarded. The formazan deposits were solubilized
by adding ethanol, and the absorption of the
solution at 560 nm was measured in a spectrophotometer.

2.4. Assessment of apoptosis

2.4.1. Feulgen reaction
Apoptosis was evaluated by morphological
analysis as previously described (Melo et al.,
2000). After 72 h of treatment, the cells were fixed
in ethanol/acetic acid (3:1), and the slides were
submitted to the Feulgen reaction and viewed at
magnification with the aid of an Olympus microscope. The apoptotic cells were defined as those
exhibiting typical characteristics as cell shrinkage,
nuclear condensation, and the formation of membrane-bound apoptotic bodies.
2.4.2. Quantification of fragmented DNA
Measurement of fragmented DNA was evaluated by the diphenylamine method in HL60 cells

219

treated for 72 h with violacein or its complexes

(Zhu et al., 1998). After the incubation period,
cells were centrifuged at 300/g for 5 min, washed
twice with ice-cold PBS, pelleted, and incubated in
200 ml of lysis buffer (10 mM Tris
/HCl, 10 mM
EDTA, pH 8.0, 0.5% Triton X-100) for 10 min at
4 8C. The lysates were centrifuged at 16 000/g
for 20 min to separate the intact chromatin (pellet)
from the DNA fragments (supernatant). The
supernatant was placed in a separate microcentrifuge tube, and pellets were resuspended in 200 ml
of lysis buffer. Both DNA fragment solution and
the intact chromatin solution were precipitated for
30 min at 4 8C in 200 ml of 1 N perchloric acid
(PCA), pelleted at 16 000 /g for 20 min and the
supernatant was removed. Pellets were resuspended in 50 ml of 1 N PCA and incubated for
15 min at 90 8C. DNA contents were determined
using the diphenylamine reagent (Zhu et al., 1998).
The percentage of DNA fragmentation was calculated as the ratio of DNA fragments to the total
DNA, the sum of DNA fragments and the intact
chromatin.

2.4.3. Caspase-2, -6, and -9 activities

Direct measurements of caspase activity were
performed using colorimetric protease kits (R&D
Systems, USA) according to the manufactures
recommendations, after the incubation of cells
with violacein or its complexes for 72 h. The assay
is based on the spectrophotometric detection of
the chromophore p -nitroanilide (pNA) after cleavage from the substrates X-p NA, where X stands
for amino acid sequences recognized by the
specific caspases VDVAD, VEID, and LEHD for
caspase-2, -6, and -9, respectively. According to
the procedure, 2 /106 cells were pelleted by
centrifugation and lysed on ice. The protein
concentration in the lysate was measured using a
Bio-Rad Protein Assay (Bio-Rad Laboratories,
Inc., USA). This assay is based on the method of
Bradford, and 200 mg of protein were incubated
with each X-p NA substrate (200 mM final concentration) at 37 8C in a microtiter plate. The
optic density of samples was measured at 405 nm.
After subtraction of the background, the increase
in the caspase activity was determined by compar-

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Fig. 2. Viability of HL60 cells after treatment with free b-CD, free violacein, violacein/b-CD (1:1), and violacein/b-CD (1:2) for 72 h.
Endpoint evaluated by MTT reduction (MTT). Each point represents the mean9/S.D. of three experiments with four replicates.

ing these results with the level of the untreated

control.

3. Results
3.1. Effects on cell viability

2.5. Statistical analysis

Data for each assay, mean9/S.D. of three
independent experiments run in four replicates,
were analyzed statistically by ANOVA. Multiple
comparisons among group mean differences were
checked with Turkey post hoc test. Differences
were considered significant when the P -value was
less than 0.05. The results were expressed as
percentages of controls and the computer software
package Origin was used to determine the IC50
values (concentration that exhibits a 50% inhibitory effect on the evaluated parameter).

The effects of violacein and its complexes on the

viability of HL60 cells were evaluated after 72 h in
culture. Using the MTT viability assay, the IC50
values for the inclusion complexes (1:1 and 1:2
violacein/b-CD molar ratios) and violacein alone
were less than 1 mM (Fig. 2). The cytotoxicity of
violacein was modified according to the molar
ratio of the complex, and it was significantly
increased by the presence of a second b-CD
molecule (IC50 /50 nM), when compared to
violacein alone (IC50 /750 nM). It is important
to mention that violacein and the inclusion complexes were less cytotoxic in V79 cells, exhibiting

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221

Table 1
Percentage of NBT reduction in HL60 cells after a 72-h treatment with free violacein, and violacein/b-CD (1:1) and violacein/b-CD
(1:2) in relation to control and free b-CD-treated cells, respectivelya
Concentrations (nM)

25
50
100
500

%NBT reduction
Violacein

Violacein/b-CD (1:1)

Violacein/b-CD (1:2)

109/5
109/3
259/5
309/7

79/3*
229/4*
309/7
339/4

209/6**
259/5**
459/7**
489/5**

a
Control experiments with the same amounts of b-CD present in the 1:1 and 1:2 inclusion complexes were carried out and used to
express the percentage of NBT reduction in violacein/b-CD-treated samples.
* P B/0.01 compared to violacein.
** P B/0.01 compared to violacein and violacein/b-CD (1:1).

an IC50 around 10 mM (de Azevedo et al., 2000a,b;

Melo et al., 2000). The b-CD had no effect on
HL60 cell viability at the concentrations used (up
to 2 mM). In such a context, Leroy-Lechat et al.
(1994) have studied the cytotoxicity of natural
cyclodextrins on P388 murine leukemic cells, and
no effects on the percentage of surviving cells were
observed up to 2 mM of b-CD.

3.2. Effects on cell differentiation

To verify the ability of violacein and its complexes to induce cell differentiation, the NBT
reduction assay was used as a marker for functional differentiation of HL60 cells. As shown in
Table 1, violacein and violacein/b-CD complexes
were able to induce NBT reduction. Moreover, a

Fig. 3. HL60 cells submitted to the Feulgen reaction. (a) Control cells; (b) 500 nM violacein-treated cells; (c) 500 nM violacein/b-CD
(1:1)-treated cells; and (d) 500 nM violacein/b-CD (1:2)-treated cells (magnification 200/).

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more pronounced effect on cell differentiation was

observed with the 1:2 inclusion complex, even at a
low concentration of violacein. At 25 nM, 20% of
HL60 cells was induced to differentiate after
incubation with the 1:2 violacein/b-CD molar ratio
complex, whereas 10% of differentiation was
achieved with free violacein, and 7% was observed
with the 1:1 molar ratio complex (Table 1).
3.3. Apoptosis induction
Since most inducers of leukemia cell differentiation may also trigger apoptosis (Kohroki et al.,
1998), we next examined whether the cell death
caused by violacein and its complexes in HL60 was
due to apoptosis. The occurrence of apoptosis was
determined by evaluation of the morphological
alterations after the Feulgen reaction. As shown in
Fig. 3, untreated cells exhibit typical non-adherent,
fairly round morphology until 72 h of culture. A
similar pattern was observed after incubation with
free b-CD even at a higher concentration of 2 mM.
Some of the treated cells still appeared normal,
whereas others exhibited dramatic morphological
alterations typical of the apoptotic process such as
chromatin condensation. In addition, numerous
apoptotic bodies, which are membrane-enclosed
vesicles that have budded off the cytoplasmatic
extension, were also observed (Fig. 3). These
apoptotic cells as well as other intact cells excluded
the trypan blue dye, suggesting that they were not
necrotizing. The percentage of DNA fragmentation after 72 h of exposure to 500 nM of all
violacein compounds was around 35%, as determined by the diphenylamine method. Taken
together, these results corroborate previous Feulgen reaction data in which apoptosis induction in
V79 cultures by free violacein was morphologically
demonstrated (Melo et al., 2000).

Fig. 4. Alterations in caspase-2, -6, and -9 activities after 72 h

of incubation with 500 nM of violacein or its b-CD inclusion
complexes in HL60 cells. Control experiments with the same
amounts of b-CD present in the 1:1 and 1:2 inclusion complexes
were carried out. Caspase activities were calculated as percentages of caspase-2, -6, and -9 activities in non-treated and free bCD samples, defined as 100%. Each column represents the
mean9/S.D. of three experiments with four replicates. *P B/
0.001 compared to control caspase-2; **P B/0.001 compared to
control and violacein caspase-2;  P B/0.001 compared to
control caspase-9; and   P B/0.001 compared to control and
violacein caspase-9.

ing the same amounts of free b-CD as those

present in the 1:1 and 1:2 inclusion complexes,
and thus they were used to express caspase
activities in violacein/bCD-treated cells. LEHDase
activity (caspase-9) increased three times over the
control after treatment with 500 nM of free
violacein. However, the activity of caspase-9 was
little changed in violacein/b-CD complexes-induced apoptosis. Assessment of the VDVADase
(caspase-2) activity showed a fourfold enhancement after the incubation of HL60 cells with 500
nM of violacein and the complexes, in relation to
the control (Fig. 4). In contrast, treatment with
500 nM of violacein, as a free drug and as
inclusion complexes, had no significant effects on
VEIDase (caspase-6) activity, compared to control
cells (Fig. 4).

3.4. Caspase activity

The effects of violacein and its inclusion complexes on the activity of caspase-2, -6, and -9 were
investigated in HL60 cells after 72 h of treatment.
The results are represented in Fig. 4. No differences were observed among control non-treated
cells and samples incubated with medium contain-

4. Discussion
Cancer is perhaps the most progressive and
devastating disease posing a threat of mortality
to the entire world despite significant advances in

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medical technology for its diagnosis and treatment. Recently, considerable attention has been
focused on identifying naturally occurring chemopreventive substances capable of inhibiting, retarding, or reversing the process of multistage
carcinogenesis. Wide arrays of indolic substances,
particularly those present in diet, microorganisms,
and medicinal plants, have been reported to
possess substantial anticarcinogenic and antimutagenic effects. The majority of these naturally
occurring indolic substances retains antioxidative
properties, which appear to contribute to their
chemopreventive activity (Dassonneville et al.,
2000).
Cultured mammalian cells provide an important
tool for evaluating the cytotoxicity of compounds
with potential therapeutic activity (Pailard et al.,
1999). The endpoints for these cytotoxic assays
often include the integrity of the cellular membranes, which provides information on the susceptibility of cellular organelles and compartments
(Rodriguez and Haun, 1999). The trypan blue
exclusion test allows evaluation of the structural
integrity of the cell membrane, whereas the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assesses mitochondrial function through the activity of succinate
dehydrogenase (Mosmann, 1983; Melo et al.,
2001). Using the MTT assay, a 50% decrease in
HL60 cell viability was found to occur upon
exposure to less than 1 mM violacein. Moreover,
viability of HL60 cells in the current study was
further decreased by the presence of cyclodextrins.
After incubation with the 1:2 violacein/b-CD
complex, the IC50 value decreased by greater
than 90% in relation to free violacein. It is
important to mention at this time that violacein
and its inclusion complexes were less cytotoxic to
V79 cells, exhibiting IC50 values around 10 mM (de
Azevedo et al., 2000a,b; Melo et al., 2000). Since
free b-CD showed no activity over the concentration range used in this study, the increase in
activity seen for the complexes compared to that
of the free violacein can be attributed to the
improved stability of violacein caused by complexation with CDs. This probably occurs because
the complexed form of violacein, which is in
equilibrium with the free violacein in solution, is

223

less prone to hydrolysis and hence serves as a

depot for continuous supply of violacein in its
active form (not published).
HL60 is a leukemic cell line blocked at the level
of promyelocytic differentiation (Verlinden et al.,
1997). Treatment perspectives, therefore, not only
include cytotoxic drugs, but more interestingly
differentiation-inducing agents. Combination of
several agents could increase their efficacy in
inducing terminal, irreversible differentiation and
growth arrest of leukemic cells, and allow for dose
reduction, thereby decreasing their side-effects
(Sokoloski and Sartorelli, 1998). Human acute
leukemia cell lines have proven particularly informative in the study of chemotherapy-associated
cell differentiation and apoptotic events. Early
studies indicated that treatment of HL60 cells
with a variety of compounds was accompanied
by changes in the expression of surface antigens
and the acquisition of a number of functions
indicative of cell differentiation into granulocytelike or monocyte/macrophage-like cells (Ujihara et
al., 1998). Since differentiated cells loose their
proliferative ability and immortality, differentiation inducers may be useful for the treatment of
leukemias (Kohroki et al., 1998; Terui et al., 1995).
The capability to reduce NBT is a first parameter
to investigate the functional differentiation. Thus,
in the present work, we evaluated the induction of
differentiation by violacein and its complexes
using the NBT assay. Our results demonstrated
that all the test compounds were able to induce
NBT reduction. Additionally, a trend to improve
the induction of cellular differentiation was observed with the complexation of violacein with bCD in a 1:2 molar ratio. These results agreed with
the increased cytotoxic effect of this complex
compared to free violacein. Considering that the
NBT assay is rather non-specific, since both
monocytes and granulocytes can stain positive,
future studies should include a more specific
determination of the cell types present in the
cultures after treatment.
Induction of programmed cell death in tumor
cells is of generous benefit for cancer chemotherapy. According to recent reports, it is possible to
postulate that some natural compounds may
regulate tumorigenesis and/or growth of cancer

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P.S. Melo et al. / Toxicology 186 (2003) 217
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cells via the induction of apoptosis in malignant

cells (Raffray and Cohen, 1997). Our results
clearly demonstrated that violacein and its complexes induced apoptosis in HL60 cells. As observed in the Feulgen reaction, treatment of these
cells with all the compounds exhibited dramatic
morphological alterations which are typical of the
apoptotic process. These include chromatin condensation and formation of numerous apoptotic
bodies. Furthermore, determination of cell viability using the vital dye trypan blue indicated that
they were not necrotizing. Apoptosis was also
demonstrated by the increase in the percentage of
fragmented DNA quantified by the diphenylamine
method.
Programmed cell death is generally associated
with activation of a number of aspartate-specific
cysteine proteases, the caspases, that are the
molecular instigators of apoptosis (Cohen, 1997).
Interestingly, whereas equal effects on caspase-2
and caspase-6 activities were found after incubation of HL60 cells with free violacein and the
complexes, a different behavior was observed in
the caspase-9 activation, because only free violacein significantly activated this caspase in our
experimental conditions. On the other hand,
treatment with violacein and its inclusion complexes similarly increased the levels of caspase-2
activity and concurrently did not affect caspase-6
activity. Activation of caspase-9 is considered to
be an important initiating event in apoptosis,
whereas caspase-2 may also be involved in the
effector phase of the process (Minko et al., 2001).
Thus, the differences observed in the caspases
activities after treatment with violacein in its free
form and complexed with cyclodextrin at both
molar ratios could be explained by the prolonged
period of incubation used in the present study.
Considering the role of caspase-2 in the effector
phase of the apoptotic event, a more pronounced
activity of this caspase might be expected, as the
exposition time of cells to the test compounds
increases. These studies further suggest the need to
evaluate the time course of caspases activities.
In summary, the present study proposes growthinhibitory effects of violacein and its b-CD inclusion complexes against HL60 cells, which are
mediated partially by the induction of cellular

differentiation and programmed cell death. In

addition, caspases seem to play an important role
in the activation of the executioner phase of the
apoptosis induction. These results provided a basis
for the potential therapeutic application of coformulation of violacein with CDs in cancer
therapy.

Acknowledgements
The Brazilian agencies CAPES, FAPESP, and
PADCT-FINEP provided financial support. The
authors would like to acknowledge Miss M.C.
Anazetti from the Departamento de Bioqumica,
UNICAMP, for her help with the diphenylamine
assay, and Mr. J.B. Fabrin Neto for his technical
contribution.

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